Preparation of micro-porous crystals and conjugates thereof

ABSTRACT

A conjugate comprising a dye-labeled microporous crystal, a stop-cock moiety, and covalently bound thereto an affinity binding agent is disclosed. The dye-labeled microporous crystal is a zeolite crystal, such as a zeolite L crystal, having a large number of channels in its interior into which the dye is loaded. The stop-cock moiety can be functionalized with an amino group to which a carboxyester group can be attached. The affinity binding agent allows for the binding to a biological moiety. The conjugate of the moiety can be used in in-vivo and/or in-vitro imaging applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to UK Patent ApplicationNo. 0621816.8 filed on 2 Nov. 2006.

FIELD OF THE INVENTION

The present invention relates to imaging of biological cells or viruses,and to modified zeolite L crystals for use in imaging of cells orviruses and for use in in-vitro diagnostics.

BACKGROUND OF THE INVENTION

Increased understanding of cellular and viral functions and regulatingmechanisms is anticipated to lead to novel approaches for therapy andnovel therapeutic agents. There is accordingly much interest in cellularand viral nano-tools as a means of investigating aspects of cellular andviral function and mechanisms. Dahm et al., (Journal of Biochemistry111:279-290, 2004) describe the use of ultra-stable zeolite particles asa tool for in-cell chemistry. In particular Dahm et al., describe theuse of labeled Y zeolite particles which were absorbed into THP-1 cellsby phagocytosis and concluded that these zeolites could be used aseffective carriers for delivery of low molecular weight molecules intocells. The zeolite particles are made ultra-stable by de-aluminating thenatural structure.

Natural zeolites are minerals that are the result of a very low-grademetamorphism, typically found in the cavities or vesicles of volcanicrock. They are framework aluminosilicate consisting of interlockingtetrahedrons of SiO₄ and AlO₄, wherein the corner-sharing SiO₄ and AlO₄tetrahedra of the crystalline aluminosilicate give rise toone-dimensional channels arranged in a hexagonal structure. Eachaluminum entity in their framework contributes a negative charge that iscompensated by an exchange of cations such as sodium, calcium and thelike that reside in the large vacant spaces and cages in the structure(see Breck in Zeolite Molecular Sieves, 752, Wiley, New York, 1974; andBaerlocher et al., in Atlas of Zeolite Framework Types, 19, Elsevier,Amsterdam, 5th edition, 2001). The stoichiometry is(K)₉[Al₉Si₂₇O₇₂].nH₂O, where n is 21 in fully hydrated materials and 16at about 22% relative humidity. Out of 9 potassium cations per unitcell, 3.6 can be exchanged by other monovalent cations, or an equivalentamount of divalent or trivalent cations.

Many forms of Zeolite are known, each form exhibiting differentgeometrical and chemical characteristics. For example, Zeolite Lexhibits one-dimensional channels running through the whole crystal,with an opening of 0.71 nm, a largest free diameter of 1.26 nm and aunit cell length of 0.75 nm (see FIG. 1 a). The centre-to centredistance between two channels is 1.84 nm. As an example a crystal with adiameter of 550 nm consists of about 80,000 parallel channels. Zeolite Lchannels can be filled with suitable organic guest molecules, althoughonly guests that can pass through the opening are able to enter thechannels. Due to the channel entrances, the chemical and physicalproperties of the base and coat of the cylindrical crystals aredifferent. Exemplary guest molecules include certain dyes possessingdesired emission properties (see Calzaferri et al., Angew Chem Int Ed42:3732, 2003). Zeolite L crystals can also be prepared in differentsizes (diameter and length). For example the length of a zeolite Lcrystal can be from 30 nm to several thousand nanometers (see Ruiz etal., Monatshefte Fur Chemie 136:77, 2005). Pure zeolite L crystals withlengths between 30 and 7000 nm have been synthesized previously(Megelski and Calzaferri, Adv Funct Mater 11:277, 2001).

Dye-loaded zeolites are described in European Patent No 1 335 879(University Bern). This patent teaches the loading of a dye intointerior channels within a zeolite L crystal. The entrances to thechannels are terminated or blocked by closure molecule, especially by aso-called stopcock moiety. The teachings of this patent document aredirected toward to the construction of a luminescent optical device, anoptical sensor device, a light-emitting device and a photonic energyharvesting device.

It is known that the channel entrances of a zeolite L crystal can beblocked or closed by a closure molecule. Such closure molecule may bechemically modified to carry for example an amino group (see Huber etal., Angew Chem Int Ed 43:6738, 2004) or a carboxylate group (H. Li, Z.Popovic, L. De Cola, G. Calzaferri, Micr Mes Mat, 95:112, 2006). In thisprocess preferably a stop-cock molecule, i.e., a molecule having a headgroup a and a tail, comprising for example a hydrophilic anchor groupand a spacer, partially enters a channel of the zeolite L crystal. Theanchor group, such as methoxysilane, and the spacer enter the channelallowing the anchor to attach to the zeolite L crystal. The bulky headgroup, e.g. a fluorenylmethylcarbamate group, remains outside thechannel entrance due to size restriction imposed by the channeldimensions. Depending on the reactivity of the label, attachment to thecrystal can either be reversible or irreversible. The head group is thenchemically detached to e.g. result in an amino functional group at theend of a channel.

Much of the work on zeolites to date has been on the use of the zeolitecrystals in photonic applications (as reported in the University of Bernpatent). The inventors of the present application have, however,realized that zeolites are used in diagnostic assays for both in-vivoand in-vitro diagnostics. The inventors have realized that it ispossible to attach an affinity binding agent e.g. via the stopcockmoiety to for a conjugate. Such conjugate comprising an affinity bindingagent under appropriate conditions will bind to another biologicalmoiety or chemical moiety. The dye loaded in the channels or at theoutside of the zeolites allows the simple detection of the conjugatebound to the biological moiety or the chemical moiety.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a) a Scanning Electron Microscopy (SEM) image of a zeoliteL crystal and (within the circle) a schematic representation of acrystal indicating that multiple strictly parallel channels arecontained within each crystal, together with a schematic representationof a longitudinal cross-section of a single channel; b) a schematiccross-section of a single channel within the zeolite L crystalfunctionalized with biphenyl terpyridine according to the invention. Theoptical microscope image shown above the schematic representation showsweak emission, with the crystals appearing as disordered single objectsor large disordered aggregates; c) a schematic cross-section of a singlechannel within two separate zeolite L crystals, each functionalized withbiphenyl terpyridine following the addition of ZnCl₂. The biphenylterpyridine groups have chelated a zinc ion, thereby joining the crystalsuch that the channel is aligned in an array according to the invention.The optical microscope image shown above the schematic representationshows a very intense emission in the blue region and the formation oflinear zeolite arrays; d) a schematic representation of a framework ofthe channels with an opening of 0.71 nm and a hexagonal symmetry; and e)a schematic representation of a side view of a channel containing a dyemolecule. The double arrow indicates the orientation of an electronictransition moment of the dye.

FIG. 2 shows a) a biphenyl terpyridine (bitpy) molecule; b) two bitpymolecules chelating a Zn²⁺ metal ion (Zn(bitpy)₂ ²⁺2(PF₆), and c) theemission spectra of bitpy, solid line (λ_(ex)=295 nm) and Zn(bitpy)₂²⁺2(PF₆)13 , dashed line (λ_(ex)=341 nm), recorded in air equilibrateddichloromethane.

FIG. 3 shows a) a confocal microscopic view of BV2 cells with zeolite Lcrystals loaded with pyronine dye; and b) an enlarged view of a BV2 cellas described in 3 a.

FIG. 4 shows amino functionalization of the whole surface of zeolite Lcrystals.

FIG. 5 shows a fluorescence microscopy image of Atto 425 functionalizedzeolite L crystals (1 μm length). The amino reactive dye Atto 425 wasattached to free amino groups which themselves were attached to thezeolite L crystal. Atto 425 NHS (NHS=N-hydroxy succinimide) is acommercially available label with a high fluorescent quantum yield of0.90. The active ester group included in the structure of the dye canreact spontaneously with the amino groups on the surface of the zeoliteL crystals.

FIG. 6 shows a) DOTA NHS (represented by circles) and tri-t-butyl DOTANHS (represented by asterisks); b) zeolite L crystals with a linkingreagent as shown in FIG. 4; and c) an amino-reactiveDOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-basedligand (DOTA NHS or tri-t-butyl DOTA NHS) attached to the linkingreagent on the zeolite L crystals.

FIG. 7 shows a) an amino-reactive DOTA-based ligand (circle), DOTA NHSor tri-t-butyl DOTA NHS (after deprotection), attached to the linkingreagent on the zeolite L crystals as shown in FIG. 6 c; and b) the DOTAfunctions on a surface of the zeolites L crystals complexed with Eu³⁺ions (represented by shaded circles).

FIG. 8 shows a fluorescence microscopy image of the DOTA functions onthe zeolites L crystals (1 μm in length) complexed with Eu³⁺ ions asshown in FIG. 7 b.

FIG. 9 shows a bifunctionalization of zeolite L crystals showing a) alaser dye (which could be any suitable cation or molecule for imagingpurpose) encapsulated into the channels, i.e. pyronine with anexcitation at 488 nm and being detected at 510 nm; and b) red emitters(which could also be any contrast agent) on the surface, i.e. ATTO 610with an excitation at 635 nm and being detected at around 650 nm.

FIG. 10 shows FTIR spectra of (a) bare zeolite L, (b) amino groupbearing zeolite L and (c) caboxyester group bearing zeolite L. Allmeasurements were performed with crystals of an average length of 30 nm.The insets contain a five time magnified section of spectra (b) and (c)respectively indicating the most relevant features. The unlabelled sharpband present in both insets is located at 1,500 cm⁻¹.

FIG. 11 shows a Raman spectrum of (a) bare zeolite L and (b)carboxyester-terminated zeolite L, both with an average crystal lengthof 30 nm.

FIG. 12 shows fluorescence microscopy images of two zeolite L crystalsmarked with TRH. The crystals have an average length of 5,000 nm. Thethin white lines are added to indicate the outline of the crystals.

FIG. 13 shows: Left—excitation (dotted) and emission (solid) spectra of30 nm Py-zeolite L and of TRH-zeolite L crystals suspended in methanol.Right - Excitation (solid) and emission (dotted) of 30 nm-sized TRH,Py-zeolite L suspended in methanol, excited at 460 nm. The excitationspectrum was detected at 660 nm.

FIG. 14 shows—Left absorption spectra of Ox1-zeolite L (solid) and ofOx1-zeolite L material functionalized to carry carboxyester groups(dotted). Right: excitation (dotted) and emission (solid) spectra ofOx1-zeolite L crystals functionalized to carry carboxyester groups. Theexcitation spectrum was observed at 690 nm and fluorescence spectrum wasexcited at 590 nm.

FIG. 15 shows—Synthesis of Ln-DOTA functionalized zeolite L crystals. Inthe first step a linker (APES) is bound to the surface of the zeolite Lcrystals via a first functional group. In the second step the chelator(DOTA) is bound to a second functional group of the linker. In the laststep the Lanthanide ions are chelated onto the surface of the zeolite Lcrystals.

FIG. 16 shows Reaction principle: (1) The amino groups located at thechannel entrances are reacted with methyl-3-isothiocyanatopropionate.(2) The terminal carboxyester groups are then coupled with acorrespondingly reactive dye.

FIG. 17 shows—Dyes used in the preparation of dye-labeled zeolite Lmaterial and their abbreviations.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a conjugate comprising adye-labeled zeolite, a stop-cock moiety, and covalently bound thereto anaffinity binding agent.

Also disclosed is a method for producing a conjugate according to thisinvention the method comprising the steps of labeling a zeolite with adye, closing the channels of the dye-labeled zeolite by a stop-cockmolecule, adding an affinity binding agent and using an appropriatecoupling chemistry to form a covalent linkage between the dye-labeledzeolite with stop-cock molecule and the affinity binding agent.

The use of a conjugate according to the present invention in variousdiagnostic procedures is also shown.

The invention further relates to a labeled zeolite loaded with animageable dye, wherein the imageable dye is selected from the groupconsisting of a metal compound that can be visualized using x-rays; acompound that can be visualized using magnetic resonance imaging (MRI);an isotope that can be imaged by x-ray computed tomography (CT),ultrasound or positron emission tomography (PET) (including photonemission computed tomography (SPECT) or other related technology).

Also described in the present invention are dye-labeled zeolithesmodified to carry a carboxyester group.

DETAILED DESCRIPTION OF THE INVENTION

Chemical modification of the zeolite crystal may direct the binding ofthe crystal to targets structures on biological moieties, such as thesurface of cells or viruses, or to targets provided inside cells thatare associated with cellular structures. When the zeolite crystals aremodified so as to bind to targets that are specifically associated withtypes of cells, viruses or cellular structures of interest, theconjugates may be used to selectively image the biological moieties.

In a first aspect of the present invention there is provided a conjugatecomprising a dye-labeled microporous crystal (such as a zeolite crystal)having a stop-cock moiety and an affinity binding agent covalently boundthereto.

The zeolite L crystal in a conjugate according to the present inventionis modified by the binding thereto of an affinity binding agent. Notwishing to be limited further, but in the interest of clarity, theaffinity binding agent may comprise any of the following; an antigen, aprotein, an antibody, biotin or biotin analogue and avidin orstreptavidin, sugar and lectin, an enzyme, a polypeptide, an aminogroup, a nucleic acid or nucleic acid analogue and complementary nucleicacid, a nucleotide, a polynucleotide, a peptide nucleic acid (PNA), apolysaccharide, a metal-ion sequestering agent, receptor agonist,receptor antagonist, or any combination thereof. For example, theaffinity binding agent can be one partner of a specific binding pair,where the other partner of said binding pair is associated with or isthe target on a cell surface or an intracellular structure. Preferablyan affinity binding agent is, a partner or member of an affinity bindingpair, or as it is also called by the skilled artisan, a partner ormember of a specific binding pair.

An affinity binding agent has at least an affinity of 10⁷ l/mol to itstarget, e.g. one member of a specific binding pair, like an antibody, tothe other member of the specific binding pair, like its antigen. Anaffinity binding agent preferably has an affinity of 10⁸ μl/mol or evenmore preferred of 10⁹ l/mol for each other.

A preferred affinity binding agent is an antibody. The term antibodyrefers to a polyclonal antibody, a monoclonal antibody, fragments ofsuch antibodies, as well as genetic constructs comprising the bindingdomain of an antibody. Any antibody fragment retaining the abovecriteria of an affinity binding agent can be used. Antibodies aregenerated by state of the art procedures, e.g., as described in Tijssen(Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) thewhole book, especially pages 43-78, Elsevier, Amsterdam).

The affinity binding agent may be directly and covalently bound to thezeolite. It may also be bound covalently via a linker group or via astopcock molecule.

The binding via a stopcock molecule allows for a well-controllablebinding of an affinity binding agent to a zeolite L crystal. Therefore apreferred aspect relates to a dye-labeled zeolite conjugated to anaffinity binding agent, wherein the affinity binding agent is bound to astop-cock molecule.

Preferably the affinity binding agent is selected from the groupconsisting of antigen and antibody, biotin or biotin analogue and avidinor streptavidin, sugar and lectin, nucleic acid or nucleic acid analogueand complementary nucleic acid and receptor and ligand. Also preferred,the conjugate according to the present invention will comprise anantigen or an antibody as an affinity binding agent.

In one aspect of the present invention the dye is located within one ormore channels of the zeolite crystal. Insertion of the dye into thechannels of the crystal may be achieved by ion exchange, particularlywhen the imageable agent is a cation, or by crystallization inclusion orby a gas phase procedure using the double or the single ampoule methodas described by Calzaferri et al, Chem. Eur. J. 2000, 6, 3456.

The zeolite crystal may be modified chemically at one or both ends,preferably adjacent the channel opening. Preferably a closure moleculeaccording to EP 1 335 879 is used modify the zeolite crystal and tosecure the dye within the zeolite crystal. Preferably the closuremolecule is a stopcock moiety which provides an advantageous means ofsecuring the dye within the zeolite crystal. Alternatively, oradditionally, such modifications may provide a means for targeting thebinding of the zeolite L crystal to cells, cellular structures orviruses.

Preferably the closure molecule is bound to one or both ends of thecrystal, in close proximity to the opening of the channel. Anappropriate closure molecule is any molecule that once bound to thecrystal at least partially physically inhibits the egress of the dyefrom within the channels of the crystal. Closure molecules preferablycontain, but are not limited to, amino, carboxylate, sugar, bioreceptor,metal ion chelating, or thiol groups.

Preferably, substantially all egress of the dye from the channels of thecrystal is prevented by a stopcock moiety. Stopcock molecules orstopcock moieties are described in detail in EP 1 335 879 the disclosurethereof is hereby explicitly included by reference. In brief, a stopcockmolecule is a molecule having a head moiety and a tail moiety, the headmoiety having a lateral extension that is larger than the channel widthand a tail moiety that has a longitudinal extension of more than thedimension of a crystal unit. The tail may comprise a spacer and ahydrophilic anchor group (called label in EP 1 335 879). The tailcomprising an anchor group, such as methoxysilane, and the spacer can atleast partially enter the channel of a zeolite L crystal. The head groupremains outside the channel. Any bulky head group, e.g. afluorenylmethylcarbamate group that remains outside the channel entrancedue to size restriction imposed by the channel dimensions can be used.

Generally it is convenient to introduce the dye into the channel of thezeolite crystal prior to modification with the stopcock moiety. Locationof the stopcock moiety, such as a chelating group, near a channelopening can assist in preventing egress of the dye from the channel. Inone aspect the present invention discloses a method for producing aconjugate as described herein above, the method comprising the steps oflabeling a zeolite with a dye, closing the channels of the dye-labeledzeolite by a closure molecule, adding an affinity binding agent andusing an appropriate coupling chemistry to form a covalent linkagebetween the dye-labeled zeolite comprising the stop-cock molecule andthe affinity binding agent.

In certain also preferred aspects the affinity binding agent maydirectly function as the closure molecule. Such conjugate between adye-labeled zeolite and an affinity binding agent will not require thepresence of a stopcock moiety.

In one aspect of the invention, a metal ion chelating group is attachedat one end of the zeolite crystal such that the entrance of a channelthrough the zeolite crystal is at least partially blocked. Preferablysuch metal ion chelating group represents the head of a stopcock moiety.

In one example, the metal ion chelating group is a terpyridinederivative. The terpyridine derivative can be biphenyl terpyridine (alsotermed “bitpy”).

Zeolite L crystals modified by biphenyl terpyridine linked to thecrystal via an amino group have been produced and characterized byfluorescence spectroscopy, since the biphenyl terpyridine exhibits anemission at around 350 nm (emission quantum yield, (Φ=0.2) and withoptical microscopy (see FIGS. 1 b and 2).

Instead, or in addition to a first modification of the zeolite beingdirected to the ends of the crystal, the external surface of the crystalmay be chemically modified in the manner discussed below.

Chemical modification of the zeolite L crystal may be preferentiallydirected to the channel entrances by controlling the ratio of moietiesto be bound to the crystal (e.g. stop cock moiety or affinity bindingagent) to the number of channel entrances. A ratio of 1:1 or less hasbeen found to favor binding of the moieties to the channel entrances. Aratio that includes more moieties than channel entrances results in amore general binding over the surface of the crystal. By first using anappropriate stopcock molecule for closing the channel entrances and forproviding a first group that may be used to attach an affinity bindingagent, the additional option becomes available to attach a second groupof interest to the side walls of a zeolite crystal. The second group ofinterest may be any moiety as required by the artisan, Preferably thesecond group of interest facilitates the solubilization of the zeoliteconjugate. Preferably said second group of interest is selected from thegroup consisting of polypeptides, sugars and polyethylene glycole. Apreferred conjugate according to the present invention thus comprisesboth, an affinity binding agent as well as a second group of interest.

The average number of channel entrances in 1 mg of zeolite L crystalscan be calculated as

n _(e) =X _(z) l _(x)×5.21×10⁻⁷ mol

X_(z)=weight of sample in mg L_(z)=average lengths of the zeolite Lcrystal in nm

The zeolite L crystals naturally carry a negative charge within theirchannel. It is preferred that modifications such as de-alumination thatreduce or eliminate the negative charge of the channel are not appliedto the crystals of the present invention.

In addition to locating the dye within one or more of the channels ofthe crystal, one or more imageable agent may be attached to the externalsurface of the microporous crystal. A chemical linker may be used tobind an imageable agent to the surface of the microporous crystal.

The dye use for labeling of a zeolite L crystal will be selected by theskilled artisan according to his needs. Any detectable label may be usedand is referred to as dye in the present invention. Preferably the dyeused for labeling the zeolite is selected from the group consisting of aluminescent compound; a fluorescent compound; a radioactive compoundthat can be visualized by photographic plates, a metal compound that canbe visualized using x-rays; a compound that can be visualized usingmagnetic resonance imaging (MRI); an isotope that can be imaged by x-raycomputed tomography (CT), ultrasound or positron emission tomography(PET) (which includes photon emission computed tomography (SPECT) orother related technology). In certain preferred aspects the dye isselected from the group consisting of a luminescent compound; afluorescent compound; and a radioactive compound.

Often the preferred dye will be a fluorescent dye. Preferably theluminescent or fluorescent dye will be selected to be capable ofentering into the channels of a zeolite L crystal. The dyes used in thepresent invention to load the microporous crystals are shown in FIG. 17.

In alternative preferred aspects the dye used in the labeling of azeolite L crystal will be a dye that can be visualized by MRI, CT orPET.

In a preferred aspect the present invention relates to the use of aconjugate according to the present invention in a diagnostic procedure.As the skilled artisan will appreciate different diagnostic procedureswill dictate different labeling and detection procedures. Preferredfields of use are for example in vitro diagnostic procedures or in vivoimaging applications, respectively.

In one preferred aspect the present invention discloses a method formeasuring an analyte by an in vitro method, the method comprising thesteps of providing a sample suspected or known to comprise the analyte,contacting said sample with a conjugate according to present inventionunder conditions appropriate for formation of an analyte conjugatecomplex, measuring the complex formed and thereby obtaining a measure ofthe analyte. The conditions appropriate for formation of ananalyte-conjugate complex need not be specified, since the skilledartisan without any inventive effort can easily identify suchappropriate incubation conditions. As the skilled artisan willappreciate there are numerous methods to measure the amount of suchanalyte-conjugate complex, all described in detail in relevant textbooks(cf., e.g., Christopoulos, T. K. (eds.), Immunoassay, Academic Press,Boston (1996)).

In a further preferred aspect the present invention discloses a methodfor in vivo imaging, the method comprising the steps of providing asample suspected or known to comprise a cell or a virus, contacting saidsample with a conjugate according to present invention under conditionsappropriate for binding of the conjugate complex to the cell or thevirus, and imaging the sample, thereby detecting the conjugate bound tothe cell or the virus.

It has also been found that one can construct a conjugate that includesone or more imageable agent bound to the surface of the microporouscrystal and having a dye held within the channel of the microporouscrystal. For example, a fluorescent or colored dye is retained withinthe channels of the zeolite L crystal and a magnetic contrast agentcoated onto the outer surface of the zeolite L crystal. Such aconstruction has the advantage that it provides the possibility for dualvisualization, for example, optically and magnetically, respectively.

The linkers mentioned above preferably have a first functional group anda second functional group at each end thereof. The first functionalgroup is capable of binding to the microporous crystal (for example,binding to the silanol groups in the microporous crystal) and the secondfunctional group is capable of binding to the required moiety (e.g. stopcock moiety, affinity binding agent or imageable agent). For example thelinker group can be an organosilane, and preferably conform to thegeneral formula R_(n)SiX_((4-n)) (wherein X is the functional groupcapable of binding to the required moiety (e.g. an alkoxy or aminogroup) and R is a non-hydrolyzable moiety). For example, the linkergroup could comprise an amino silane (such as(3-aminopropyl)triethoxysilane) and a DOTA(1,4,7,10-tetraazacyclodedecane-1,4,7,10-tetraacetic acid). Theaminosilane may be bound to the crystal via its ethoxysilane groups, andto the DOTA via its amine groups after activation of DOTA by NHS. Such alinker is capable of chelating imageable agents such as gadolinium andeuropium.

In one aspect of the present invention, the conjugates are capable ofbeing taken up by a cell, such as a biological cell. The microporouscrystal can, therefore, be located in the cytoplasm of a cell. Thetaking up of the conjugates can occur via phagocytosis. It has beenfound that in order to optimize the taking up of the conjugate in acell, the microporous crystals of the conjugate preferably have adiameter of 100 nm or less. Additionally, it is preferred that themicroporous crystals do not bind to each other or form agglomerations,but instead are provided as discrete crystals. The taking up of theconjugate in the cell allows the use of the microporous crystal inin-vivo measurement techniques.

Solubilization and specific uptake of the zeolites into a cell can bealso achieved by selective functionalization of the surface and/orchannel entrances of the zeolite L crystal.

Alternatively, or in addition to being taken up by a cell, the conjugatemay be bound to the surface of a cell or virus.

In a further aspect of the present invention, there is provided a methodfor imaging cells, viruses or cellular structures, comprising the stepof incubating cells or viruses with a conjugate comprising themicroporous crystals and the dye and, optionally, the one or moreimageable agent, such that the conjugate becomes physically associatedwith the cells, viruses, or cellular structures within the cells.Preferably the conjugate is any one, or any combination, of theconjugates described in the first aspect of the present invention.

The method may further comprise the step of visualizing the dye and,optionally, the other imageable agent after physical association of theconjugate and the cells, viruses, or cellular structures within thecells. The nature of the visualization step is dictated by the type ofimageable agent used. For example, when imageable agent is a dye thestep of visualization is carried out optically, when imageable agent isa gadolinium the step of visualization can be carried out using MagneticResonance Imaging (MRI) or Functional Magnetic Resonance Imaging.

The biological moieties may be incubated with a conjugate according tothe present invention in a suspension. Preferably the cells and virusesare suspended in a physiological acceptable buffer (e.g. PBS). Theconjugate and the biological moieties are preferably incubated togetherto allow for binding of the conjugate to the biological moiety.Incubation may take from 1 to 20 minutes (preferably about 5 minutes),and preferably is carried out at a temperature that is physiologicallyacceptable to the cells or viruses (e.g. about 37° C.). Prior toincubation of the conjugate with the biological moiety the conjugate maybe sonicated in water to reduce aggregation of the conjugate.

Generally the cells will be alive, and the viruses viable, respectively,during and immediately subsequent to the taking up of the microporouscrystal, or binding of the microporous crystal to the surface of thecell or virus. Suitable cells include eukaryotic cells, especiallyanimal cells (such as mammalian cells), plant cells and insect cells.

Radiationless excitation energy transfer from the dyes encapsulated inthe channels of a microporous crystal to acceptors covalently bound toits external surface and then further through a thin insulating layer toa semiconductor has been demonstrated by S. Huber and G. CalzaferriChem. Phys. Chem. 5 (2004) 239. A general approach for interfacing thedye loaded microporous crystals to their surroundings involves theaddition of stopcock molecules. Employing a multi-step reactionprinciple, one can synthesize zeolite crystals functionalized with freeamino groups located only at the channel entrances, as discussed above.This resulting material can be used as a precursor to which any aminoreactive substance can, in principle, be bound.

The possibility of using carboxyester functionalization in addition tothe amino functionalization widens the scope of chemical compounds orbiological moieties that can be attached to the microporous crystals.The approach followed to synthesize such materials is sketched in FIG.16. In a preferred aspect the present invention relates to acarboxyester functionalized zeolite L. Also preferred said carboxyesterfunctionalized zeolite L is labeled with a dye. Preferably the dye usedin the labeling of a carboxyester functionalized zeolite L is selectedfrom the group consisting of a light absorbing compound, a fluorescentcompound; a luminescent compound; a radioactive compound that can bevisualized by photographic plates, a metal compound that can bevisualized using x-rays; a compound that can be visualized usingmagnetic resonance imaging (MRI); an isotope that can be imaged by x-raycomputed tomography (CT), ultrasound or positron emission tomography(PET) (which includes photon emission computed tomography (SPECT) orother related technology).

In one aspect the synthesis of a carboxyester functionalized zeolite Lis carried out in two steps, in a first step (1) the amino-modifiedzeolite L crystals are prepared by means of the procedure describe in S.Huber and G. Calzaferri Angew. Chem. Int. Ed. 43 (2004) 6738. In thesecond phase (2) the free amino group attached to the entrance of eachchannel is reacted with the thiourea group frommethyl-3-isothiocyanatopropionate according to a reaction described T.Phuong, T. Khac-Minh, N. Thi Van Hag, Thi Ngoc Phuong Bioorg. Med. Chem.Lett. 14 (2004) 653. With appropriate silanes containing a carboxylestergroup a one step procedure for providing a carboxylester functionalizedzeolite crystal is also possible.

The inventors have also found that zeolite L crystals can be associatedwith an imageable agent and used for imaging of cells or viruses, suchas for in vivo and in vitro imaging purposes and as label for in vitrodiagnostics. In a preferred aspect the present invention thereforerelates to a labeled zeolite loaded with an imageable dye, wherein theimageable dye is selected from the group consisting of a metal compoundthat can be visualized using x-rays; a compound that can be visualizedusing magnetic resonance imaging (MRI); an isotope that can be imaged byx-ray computed tomography (CT), ultrasound or positron emissiontomography (PET) (which includes photon emission computed tomography(SPECT) or other related technology). Also preferred is dye-labeledzeolite L wherein said dye is a dye that can be visualized by MRI, CT orPET.

The zeolites of the invention can be used for molecular imaging.Molecular imaging aims at visualizing molecules or molecular eventsoccurring at cellular level which are the “signatures” of a givendisease and allow for an early diagnosis of a disease and the efficientmonitoring of therapeutic treatments. Among the available imagingmodalities, much attention has been devoted to Magnetic ResonanceImaging (MRI) thanks to the anatomical resolution that can be attainedin its images. However MRI is a relatively insensitive modality andrequires large amounts of proton relaxation agents to affect thecontrast naturally occurring between different anatomical regions. Inorder to get a 50% contrast enhancement in an MR image with thecurrently available commercial contrast agents, one needs to reach alocal concentration of ca 50 μM i.e. a number of e.g. ca. 10⁹ Gd centersper cell. [see, for example, Aime et al, S. Magnetic Resonance Imaging16(4): 394-406 (2002).]

The development of MR-Molecular Imaging applications necessitatesfurther tools that allow the delivery of a large number of imagingreporting units at the targeting site. For instance, Lanza et al.(Magnetic Resonance in Medicine 53(3): 621-627 (2005)) reported thevisualization of integrin receptors (which overexpression characterizesthe tumor endothelium) by using functionalized microemulsion particlesloaded with 105 Gd(III) complexes.

There is furthermore an active search of carriers that can suitablytarget the diseased cells with their payload of imaging reporters. Muchattention is currently devoted to phospholipid based systems such asliposomes (see, for example, Mulder W J M, Strijkers G J, Griffioen A W,van Bloois L, Molema G, Storm G, Koning G A, Nicolay K, BioconjugateChemistry 15 (4): 799-806 2004 and Mulder W J M, Douma K, Koning G A,Van Zandvoort M A, Lutgens E, Daemen M J, Nicolay K, Strijkers G J,Magnetic Resonance in Medicine 55 (5): 1170-1174 2006) and micelles(see, for example, Accardo A, Tesauro D, Roscigno P, Gianolio E, PaduanoL, D'Errico G, Pedone C, Morelli G Physicochemical properties of mixedmicellar aggregates containing CCK peptides and Gd complexes designed astumor specific contrast agents in MRI, Journal of the American ChemicalSociety 126 (10): 3097-3107 2004). The systems of these publications arereadily taken up by the cells as the phospholipids are the mainconstituents of cell's membrane. It is of interest to design rigidparticles to tackle applications not feasible with phospholipids basedsystems. Moreover, the particles may act themselves as carriers ofadditional imaging probes to add further physiological information tothe high spatial resolution delineated by MRI contrast agents.

As is discussed above, the components responsible for imaging can beentrapped inside the channels of the microporous crystals (for instance,Gd-ions or any derivatives) or are surface groups on the surfaces of themicroporous crystals. The components incorporate either the red emittingEu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) orMRI contrast agent Gd-DOTA moiety. Ln-DOTA chelates have been chosen dueto their high stability and therefore low toxicity. The fact that theDOTA is coordinating the lanthanide ion the possible use of this systemas an MRI contrast agent. Since Gd(III) is toxic in the free ionic formit has to be chelated with proper ligands providing extremely highbinding constants such as DOTA {DTPA}. The toxicity of Gd (III) is thennegligible. Also the synthetic ease of changing the metal ion makesthese systems very versatile materials, allowing the rich physicalproperties of all the lanthanides to be exploited. The inorganicscaffolds to which the Ln-DOTA is coupled are Zeolite L crystals.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

The present invention will now be further described with reference tothe following, non-limiting examples.

EXAMPLES Example 1 Zeolite Modification

The cylindrical zeolite L crystals used in this example were of meanlength of 2.2 μm and mean diameter of 1.2 μm.

Terpyridine Ligand Terminated Zeolite L Crystals

Amino terminated zeolite L crystals were prepared (according to Huber etal., Angew Chem Int Ed 43: 6738, 2004) by suspending zeolite L crystalsin n-hexane, and calculating the amount of channel entrances as follows:number of zeolite L channel entrances: n_(e)=(X_(z)/l_(z))×5.21×10⁻⁷(ne: number of channel entrances in mols, X_(z): mass of zeolite in mg,I_(z): average length of zeolite in nm). 9-fluorenylmethyl carbamateN-hydroxysuccinimidyl ester was reacted with(3-aminopropyl)methoxydimethylsilane and the reaction product was addedto the zeolite L suspension, equalling the amount of channel entrances.The suspension was sonicated (20 minutes) and heated at reflux for 3hours followed by centrifugation. The modified zeolite L crystals weresuspended in dry DMF (N,N′-dimethylformamide) containing 20%(volume/volume) piperidine, stirred for 1 hour and washed with methanol,resulting in amino terminated zeolite L crystals as shown in FIG. 1.Around 40 mg of amino terminated zeolite L crystals were suspended in 2ml of dry DMF and mixed with 10 ml of triethanolamine. The suspensionwas heated to 65° C. in a sealed glass tube. Activated terpyridineligand (TpyPh₂COOSu) (100 fold excess calculated with respect to theabove formula) was suspended in 1 ml of dry DMF and half of solution wasadded to the warm suspension. After stirring for 15 minutes theremaining amount was added and solution stirred for additional 2 hours.Centrifugation was performed from DMF and from methanol yielding theterpyridine ligand terminated zeolite L crystals that were further driedat 60° C. in an oven for 12 hours. The results are shown in FIG. 1 b.

Example 2 Modification of Zeolite L Crystals of 30 nm

Prior to the functionalization of the channel entrances as described inExample 1, the zeolite L crystals of 30 nm were pretreated as follows:

25.6 mg of zeolite L crystals were suspended in 2.8 ml of an aqueous 0.1M KNO₃ solution. The suspension was sonicated for 10 minutes, stirredfor 2 hours at 60° C., centrifuged, and the supernatant was discarded.After twice washing steps with bidistilled water, the zeolite L crystalswere dried at 65° C. overnight. After treatment with citrate buffer andequilibration in 22% humidity atmosphere, the weight of the zeolite Lcrystals was determined, the number of channel entrances was calculatedaccording to the formula disclosed in example 1, and the zeolite Lcrystals were modified as described in Example 1.

Example 3 Zeolite L Crystals Assembly with Zn²⁺

Around 10 mg of the ligand-terminated zeolite L crystals prepared asdescribed in Example 1 were suspended in 1 ml of methanol and warmed to60° C. A calculated amount (2 eq of zeolite L channel entrances: 1 eq ofZnCl₂; calculated as described in Example 1) of the standard solution ofZnCl₂ in methanol was added slowly to the stirred suspensions. Thereaction mixture was sonicated for 1 minute and stirred for 1.5 days.

Centrifugation was performed from methanol yielding the assembledzeolite L crystals that were further dried at 60 ″C in an oven for 2hours. The results are shown in FIG. 1 c.

Example 4 Zeolite L Crystals for in Vivo Imaging

Zeolite L crystals were loaded with pyronine dye via an ion exchangeprocedure according to Calzaferri et al. (Angew Chem Int Ed42:3732-3758, 2003). A solution of the dye was added to a zeolite Lcrystal and the mixture was stirred resulting in incorporation of thedye molecules into the channels of the zeolite L crystal. Aftercentrifugation of the suspension, the supernatant was discarded and thezeolite L crystals were rinsed to remove remaining unincorporated dyemolecules. After a further centrifugation step, the supernatant wasdiscarded resulting in dye loaded zeolite L crystals.

1 mg of the zeolite L crystals were mixed with 1 ml of bidestilled waterand sonicated for 30 minutes. 2 μl of the sonicated zeolite L solutionwas added to 200 μl of a BV2 cell suspension, containing 250 000 cells,in PBS buffer.

The suspension was diluted to 400 μl with PBS buffer. The suspension wasincubated for 5 min at 37° C. After incubation, a 10 μl aliquot of thesuspension was placed on a glass slide and microscopically checked (seeFIG. 3).

Example 5 Amino Functionalization of Surfaces of Zeolite L Crystals of 1μm

40 mg of zeolite L crystals (1 mm) were suspended in dry toluene (4 ml)and 30 μl of (3-aminopropyl)triethoxysilane (APES) were added. Thesuspension was sonicated (30 seconds) and stirred for 4 hours at 105° C.

After cooling down to room temperature the sample was centrifuged andthe supernatant was discarded. The sample was washed twice with toluene,resulting in amino functionalized surfaces of the zeolite L crystals.

Example 6 Amino Functionalization of Surfaces of Zeolite L Crystals of30 nm

As described in Example 5, 40 mg of zeolite L crystals (30 nm) weresuspended in dry toluene (4 ml) and 30 μl of(3-aminopropyl)triethoxysilane (APES) were added. The suspension wassonicated (30 seconds) and stirred for 4 hours at 105° C.

After cooling down to room temperature the solvent was removed byevaporation, the sample was then transferred to a centrifuge tube usingEt₂O and then centrifuged (30 minutes, 4000 rpm). The supernatant wasdiscarded, the sample was washed with MeOH once and dried at 75° C.overnight, resulting in amino functionalized surfaces of the zeolite Lcrystals.

Example 7 Attaching DOTA-NHS to Amino Functionalised Zeolite L Crystalsof 1 μm

4 mg of amino functionalized zeolite L crystals, as described in Example5, and 6 mg (0.0072 mmol) of DOTA-NHS were suspended in 1 ml dry DMF and16 μl of DIPEA (diisopropylethylamine) were added. After stirringovernight, the solvent was removed by evaporation and the DOTA modifiedzeolite L crystals were suspended in approximately 2 ml H₂O.

Example 8 Attaching DOTA-NHS to Amino Functionalized Zeolite L Crystalsof 30 nm

30 mg of amino functionalized zeolite L crystals, as described inExample 6, and 94 mg (0.113 mmol) of DOTA-NHS were suspended in 1 ml dryDMF and 150 μl of DIPEA were added. After stirring overnight, thezeolite L crystals completely solubilized and were precipitatedafterwards by addition of 5 ml Et₂O. Following centrifugation, the DOTAmodified zeolite L crystals were washed with Et₂O, twice, and driedovernight.

Example 9 Attaching Tri-t-butyl DOTA-NHS to Amino Functionalized ZeoliteL Crystals of 1 μm

2 ml (max. 0.013 mmol) of DOTA activated ester solution in DMSO(Tri-t-butyl DOTA-NHS) were added to 4 mg of amino functionalizedzeolite L crystals, derived as described in Example 5. 16 μl DIPEA wereadded, and after stirring overnight, the solvent was removed. Afterwashing the Tri-t-butyl DOTA modified zeolite L crystals once with H₂O,they were dried overnight.

Example 10 Deprotection of Tri-t-butyl DOTA Modified Zeolite L Crystals

The dried Tri-t-butyl DOTA modified zeolite L crystals from Example 9were suspended in 5 ml of CH₂Cl₂, and 5 ml TFA were added drop wise. Thesuspension was stirred overnight at room temperature. After removal ofthe solvent under reduced pressure the acid was removed by addition andevaporation of successive portions of DCM (2×10 ml), MeOH (2×10 ml) andthen ether (2×10 ml), resulting in DOTA modified zeolite L crystals.

Example 11 Complexation of Eu(III) Ions with DOTA Modified Zeolite LCrystals

5.2 mg of EuCl₃.H₂O (0.014 mmol) were added to the DOTA modified zeoliteL crystals as described in Examples 7 and 10, re-suspended in H₂O. 3drops of 1 N NaOH were added and the suspension was stirred overnight.After centrifugation the supernatant was removed and Eu-modified zeoliteL crystals were washed 10 times with EtOH until the washing solution didnot show a europium signal in the emission spectroscopy after additionof a sensitizer.

Example 12 Complexation of Gadolinium Ions with DOTA Modified Zeolite LCrystals

The complexation of DOTA modified zeolite L crystals was performed asdescribed in Example 11. Instead of EuCl₃.H₂O, 54.9 mg of GdCl₃.6H₂O(0.148 mmol) were added. After washing, the Gd-modified zeolite Lcrystals were dried for several days at 75° C.

Example 13 Bifunctionalization of Zeolite L

The zeolite L have been filled with a fluorescent dye, pyronine,following above described procedure and then functionalized on thesurface with DOTA ligands (see above) which have been then complexedwith Eu(III) ions. The resulted bifunctional system was washed withethanol and dried. Analysis at the confocal microscope reveals a doubleemission due to both the pyronine and Eu complex, as shown in FIGS. 9 aand b.

Example 14 Carboxyester Functionalization of Zeolite L Materials andReactions

Zeolite L materials were synthesized as described in A. Zabala Ruiz, D.Brühwiler, T. Ban, G. Calzaferri, Monatshefte für Chemie 136 (2005) 77.Py was synthesized and purified according to the procedure given in H.Mas, A. k Khatyr, G. Calzaferri, Micropor. Mesopor. Mater 65 (2203),233. Ox1 and TRH were obtained from Molecular Probes/Invitrogen and usedwithout further purification. Dye-loaded zeolite L samples were preparedaccording to an ion-exchange process previously described in A. Devaux,Z. Popovic, O. Bossart, L. De Cola, A. Kunzmann, G. Calzaferri,Micropor. Mespor. Mater. 90 (2006) 69 and G. Calzaferri, S. Huber, H.Maas, C. Minkowski, Angew. Chem. Int. Ed. 42 (2003) 3732 and C.Minkowski, G. Calzaferri, Angew. Chem. Int. Ed. 44 (2005) 5325. Aminogroup modified zeolite L crystals were prepared according to theprocedure published in S. Huber, G. Calzaferri, Angew. Chem. Int. Ed. 43(2004) 239 after first treating the zeolite in citrate buffer of pH 5for 1 h. The attachment of carboxyester terminated groups to the channelentrances of zeolite L was done by reacting the amino group modifiedmaterial with methyl-3-isothiocyanatopropionate by means of a reactiondescribed in T. Phoung, T. Khac-Minh, N. Thi Van Ha, H. Thi Ngoc Phuong,Bioorg. Med. Chem. Lett. 14 (2004) 653. The coupling of Texas redhydrazide (TRH) to the carboxyester functionalised zeolite L was carriedout as follows: 10 mg of the precursor material was suspended in 2 mL ofmethanol and the suspension was sonicated for 10 min. Texas redhydrazide (3-4 molar equivalent relative to the number of channelentrances) dissolved in 1 mL of methanol was added dropwise to thezeolite L suspension and the suspension was stirred at 330 K for 4 h.The reaction product was washed several times with methanol and thendried at 353 K overnight.

Physical Measurements

FTIR spectra were recorded as KBr disks on a PerkinElmer FTIR spectrumone, with a resolution of 4 cm−1. The FT-Raman spectra were measuredwith the Raman accessory of a BOMEM DA8. This spectrometer was equippedwith a liquid nitrogen cooled InGaAs detector and a quartz beamsplitter. A continuous wave, diode pumped Nd3+; YAG laser (CoherentCompass 10642500MN) was used as exciting beam. Rayleigh scattering wasremoved by two holographic super notch filters (Kaiser Optical SystemsHSPF-I064.0-1.0) in 0° position. All Raman spectra were measured with aresolution of 16 cm−1. Duran glass capillaries served as sample holders.Fluorescence spectra were measured with an LS 50 B Perkin-Elmerluminescence spectrophotometer and absorption spectra with the Lambda900 Perkin-Elmer UV/Vis/NIR instrument. Fluorescence microscopy imageswere recorded on an Olympus BX 60 microscope.

Results

The synthetic procedure was carried out with zeolite L crystals of anaverage length of 30 nm and 5000 nm. The choice of two extreme lengthsdemonstrates that the procedure is independent of the crystal size andalso allows the characterization of the material with differenttechniques. A first and easy test is to measure the infrared and Ramanspectra of the functionalized material. This was done with 30 nmcrystals, because the concentration of substituents is higher due to theincreased surface to volume ratio.

The corresponding FTIR spectra are given in FIG. 10 a-b. The broad,intense band and the peak centered at about 1640 cm⁻¹ in all spectra isdue to the presence of water in the samples. The band at 1570 cm⁻¹ inFIG. 10( b) can be attributed the N—H valence angle deformation of theamino group. This band disappears in FIG. 10( c) and is replaced by anew band at lower frequency (1546 cm⁻¹) which indicates a successfulreaction. Furthermore, the presence of the C═O stretching vibrationarising from the ester group at 1720 cm⁻¹ can easily be recognized. Thespectra in FIGS. 10( b) and 10(c) exhibit absorption bands in the regionfrom 2980 cm⁻¹ to 2830 cm⁻¹ which are due to —CHz- and —CH₃— groups. Adetailed comparison of the bands and there assignment is reported inTable 1.

FIG. 11 shows the Raman spectrum of carboxyester terminated and of barezeolite L crystals. The average crystallite length in each case was 30nm. The low intensity of the spectra is not only due to the fact thatzeolite L is a very weak Raman scatterer, but also to the lowconcentration of substituents (which are only present at the channelentrances). The Raman spectrum of the bare zeolite L contains only oneclear band at 500 cm⁻¹, that can be assigned to 0-T-0 (T=Si or AI)bending vibration. The spectrum of the modified sample exhibits someadditional bands: the one at 732 cm⁻¹ is due to the N—H wagging of thesecondary aliphatic amines while the peak at 790 cm⁻¹ can be attributedto the Si—C stretching mode. The weaker band around 1001 cm⁻¹ arisesfrom the C═S stretching vibration. The last two peaks at 1310 cm⁻¹ and1452 cm⁻¹ were assigned the —CHz- twisting motion and the Si—CH₃asymmetric stretching modes.

The vibrational spectra tell us that the reaction was successful. Inorder to get information on the spatial distribution of the attachedfunctional groups, the larger crystals were investigated by means ofoptical microscopy after marking the ester groups on the zeolite Lsurface with a fluorescing dye. For this investigation, 5000 nm longcrystals were labeled with the carboxy reactive dye TRH. The couplingreaction was carried out in a similar way as described in T. Phuong, T.Khac-Minh, N. Thi Van Ha, H. Thi Ngoc Phuong, Bioorg. Med. Chem. Lett 14(2204) 653. The resulting fluorescence microscopy image in FIG. 12 showsthat the dyes, and therefore the carboxy groups, are preferentiallylocated at the channel entrances of the crystals. This interpretation iscorroborated by optical microscopy images of crystals that were modifiedover the whole surface. Such images are reported in FIG. 3 of S. Huberand G. Calzaferri, Angew. Chem. Int. Ed 43 (2004) 6738.

An independent way to test whether the carboxyester reactive dye isfixed to the zeolite L surface is to perform energy transferexperiments. Py and TRH are suitable donor and acceptor because of thefavorable spectral overlap between the fluorescence spectrum of Py andthe absorption spectrum of TRH. Another advantage is that thefluorescence maximum of the acceptor TRH (612 nm) is well separated fromthat of Py (510 nm). The loaded 30 nm sized zeolite L was loaded withPy, to a loading level of about 10%. The Py-loaded zeolite L was thenmodified with TRH. Excitation and fluorescence spectra of 30 um longzeolite L crystals loaded with only Py or TRH, suspended in methanol,are shown in the left part of FIG. 13. Similar spectra for thePy-zeolite L material with TRH modified channel entrances, that weremeasured under the same conditions as above, are displayed on the rightside of FIG. 13. The emission spectrum of this sample was recorded byexciting the sample at 460 nm where only Py absorbs light. The emissionspectrum consists of two bands. The first emission band around 510 nmcan be assigned to Py, while the second emission band at 612 nmcorresponds to TRH. Since TRH cannot be excited directly at 460 nm, thesecond emission band is due to an effective energy transfer from Py toTRH. The excitation spectrum of this mixed material, detected at 660 nm,corresponds neatly to the superposition of the excitation curves of eachseparate dye. Such behavior is to be expected in the case of resonanceenergy transfer.

While carrying out these experiments, it was observed that themodification of zeolite L at the channel ends with carboxyester groupsleads to a better dispersability of the crystals in toluene. In order tofurther investigate this aspect, 30 nm sized Zeolite L samples wereloaded with Ox1 and then modified their channel entrances withcarboxyester groups. The absorption, fluorescence, and excitationspectra of the prepared material suspended in toluene are shown in FIG.14. The absorption spectra of the carboxyester functionalized and of theunsubstituted Ox1-zeolite L sample (depicted in the left panel of FIG.14) show a well resolved band belonging to Ox1. It is also interestingto notice that the baseline in the range from 500 nm to 350 nm of thecarboxyester terminated material is much lower compared to that of anunmodified sample. As already mentioned, the surface modificationimproves the dispersability of the zeolite L crystals by preventingtheir aggregation. The resulting smaller particle size leads to areduction in the Rayleigh scattering intensity. The improveddispersability of the treated zeolite material can also be observed byinvestigating a droplet of the suspension under an optical microscope.Comparable observations on other surface treated zeolite L nanoparticleswere published in A. Devaux, Z. Popovic, O. Bossart, L. De Cola, A.Kunzmann, G. Calzaferri, Micropor. Mesopor. Mater. 90 (2006) 69. Theshape of the bands in the luminescence spectra of carboxyesterterminated Ox1-zeolite L (see FIG. 14, right side) is of good qualityand they occur at the expected wavelengths. Thus proving that thefunctionalization procedure used in this study has no detrimental effecton the dyes inserted into the zeolite L channel.

Example 15 Zeolite L Crystals as Probes for MRI Applications Synthesis

The ligand DOTA NHS was obtained from Macrocyclics (USA) and used asreceived. All other reagents were obtained from Sigma-Aldrich and usedas received. Pure zeolite L crystals of 1 μm and 30 μm were synthesizedand characterized as described (Megelski, Calzaferri, Adv. Funct. Matter(2001) 277 and Zabala Ruiz, Brühwiler, Ban Calaferri, Monatshefte fürChemie, 136 (2005), 77). The potassium exchanged form was used for allexperiments.

Amino Functionalization of Zeolite L 1 μm

40 mg of zeolite L crystals 1 μm were suspended in dry toluene (4 mL)and 30 μL of (3-aminopropyl)triethoxysilane (APES) were added. Thesuspension was sonicated for 30 s and stirred for 4 h at 105° C. Aftercooling down to room temperature the sample was centrifuged and theoverstanding solution was pipetted off. The sample was washed twice withtoluene.

Amino Functionalization of Zeolite L 30 nm

40 mg of zeolite L crystals 30 nm were suspended in dry toluene (2 mL)and 30 μL of (3-aminopropyl)triethoxysilane (APES) were added. Thesuspension was sonicated for 30 s and stirred for 4 h at 105° C. Aftercooling down to room temperature Et₂O (6 mL) was added and the samplewas centrifuged for 30 min at 4000 rpm. After pipetting the overstandingsolution off, the sample was washed with MeOH once and then dried in theoven at 75° C. overnight.

Reaction of NH₂ Groups Attached on the Whole Surface of the Zeolite L 1nm with Atto-NHS

1 mg zeolite L crystals and 0.5 mg (0.01 mmol) Atto-NHS were suspendedin 2 mL dry DCM. The suspension was stirred overnight at roomtemperature. After centrifugation the sample was washed with DCM andMeOH (5×3 mL) until no Atto emission was seen in the washing solution.

Reaction of NH₂ Groups Attached on the Whole Surface of the Zeolite L 30nm with Atto-NHS

1 mg zeolite L crystals and 0.5 mg (0.01 mmol) Atto-NHS were suspendedin 2 mL dry DCM. The suspension was stirred overnight at roomtemperature. After centrifugation the sample was washed with DCM andMeOH (6×3 mL) until no Atto emission was seen in the washing solution.

Reaction of NH₂ Groups on the Surface of Zeolite L 1 □m with DOTA-NHS

4 mg of zeolite L crystals and 6 mg (0.0072 mmol) of DOTA-NHS weresuspended in 1 mL dry DMF and 16 μL of DIPEA were added. After stirringovernight the sample was transferred into a round bottom flask and thesolvent was removed on the rotary evaporator. Afterwards the sample wastransferred back into a centrifuge tube, using H₂O (ca. 2 mL) assolvent.

Reaction of NH₂ Groups on the Surface of Zeolite L 30 nm with DOTA-NHS

30 mg of zeolite L crystals and 94 mg (0.113 mmol) of DOTA-NHS weresuspended in 1 mL dry DMF and 150 μL of DIPEA were added. After stirringovernight the crystals solubilized. After addition of 5 mL Et₂O thesample precipitated and was centrifuged. The sample was washed with Et₂Otwice and dried in the oven overnight.

Complexation of Eu(III) Ions with DOTA at the Zeolite L Crystals

To the DOTA functionalized zeolite L samples in H₂O 5.2 mg of EuCl₃.6H₂O(0.014 mmol) were added. After addition of 3 drops of 1 N NaOH themixture was stirred overnight. After centrifugation and removal of theoverstanding solution the sample was washed 10 times with EtOH until thewashing solution did not show a europium signal in the emissionspectroscopy after addition of a sensitizer.

Complexation of Gd(III) Ions with DOTA at the Zeolite L Crystals

The DOTA functionalized zeolite L sample was suspended in 2 mL H₂O and54.9 mg of GdCl3.6 H₂O (0.148 mmol) were added. After addition of a fewdrops of 1 N NaOH the mixture was stirred overnight. Aftercentrifugation and removal of the overstanding solution the sample waswashed with EtOH. The sample was then washed with EtOH and in the ovenat 75° C. Eventual excess of uncomplexed Gd(III) ions has been removedby 24 h dialisis of the Gd-DOTA-zeolite L system against water.

Results

Steady-state emission spectra were recorded on a Spex Fluorolog 1681equipped with a Xe arc lamp, a Hamamatsu R928 photomultiplier tube anddouble excitation and emission monochromators. Emission spectra werecorrected for source intensity and detector response by standardcorrection curves. The emission was detected at a right angle.Fluorescence microscopy images were taken of zeolite L crystals having alength of about 1 μm. Fluorescence microscopy was performed with anOlympus BX 41 microscope equipped with an Hg high pressure lamp, and theappropriate filters. Scanning electron microscopy was performed with aLEO 1530 VP.

The longitudinal water proton relaxation rate was measured by using aStelar Spinmaster (Stelar, Mede, Pavia, Italy) spectrometer operating at20 MHz, by mean of the standard inversion-recovery technique. Thetemperature was controlled with a Stelar VTC-91 air-flow heater equippedwith a copper constantan thermocouple (uncertainty 0.1° C.). The proton1/T1 NMRD profiles were measured over a continuum of magnetic fieldstrength from 0.00024 to 0.47 T (corresponding to 0.01-20 MHz protonLarmor Frequency) on a Stelar field-cycling relaxometer. The relaxometerworks under complete computer control with an absolute uncertainty in1/T1 of ±1%. Data points from 0.47 T (20 MHz) to 1.7 T (70 MHz) werecollected on a Stelar Spinmaster spectrometer working at variable field.The Gd(III) concentration of Gd-DOTA-zeoliteL solutions, for therelaxometric characterization, was determined mineralizing a givenquantity of sample solution by the addition of HCl 37% at 120° C.overnight: from the measure of the observed relaxation rate (R1obs) ofthe acidic solution, knowing the relaxivity (r1p) of Gd(III) aquaion inacidic conditions (13.5 mM−1 s-1), it was possible to calculate theexact Gd(III) concentration (this method was calibrated using standardICP solutions, and the accuracy was determined to be 1%).

A dual probe in which the known filled zeolite L is functionalized onthe entire surface using chelating ligands able to strongly bindluminescent or paramagnetic ions was designated. In one aspect of theinvention empty zeolite L crystals with a length of about 1 μm wereused. These larger systems have similar structural and chemicalproperties to those of 30 nm length, but are of course easy tocharacterize with different techniques.

The first step in the reaction involves functionalizing the wholesurface of the zeolite with functional groups which could then bereacted with chelating systems. 3-aminopropyl triethoxysilane (APES) isknown to react with free Si—OH groups on the zeolite surfaceleading to agood coverage of the surface according to the simplified scheme depictedin FIG. 15.

Example 16 Zeolite L Crystals as Labels for In Vitro DiagnosticApplications Dye Loading of Zeolite L with Cationic Dyes

Zeolite L material is synthesized and characterized as described (E.g.S. Megelski, G. Calzaferri Adv. Funct. Mater. 2001, 11, 277.b)

For the loading with cationic dyes, the potassium-exchanged form of thecrystals is used. As a dye, laser grade Oxazine 1 is used withoutfurther purification. The Ox 1 is inserted into the zeolite L channelsby ion exchange out of toluene. Typically the zeolite L (20 mg) issuspended in toluene (5 ml). The suspension is stirred vigorously whilethe desired amount of Ox 1 suspended in toluene (100 ml) is added. Themixture is heated to 80° C. for a few minutes. To remove any dyemolecules adsorbed on the outer surface of the zeolite, the crystals arethen washed with diluted Genapol X-080 from Fluka. Typically a wateryGenapol X-080 solution (1:500; 5 ml) is added and the resultingsuspension is sonicated for 10 mm. It is then centrifuged until thesupernatant is clear and can be discarded.

Example 17 Dye Loading of Zeolite L with Neutral Dyes

Zeolite L material is synthesized and characterized as described (E.g.S. Megelski, G. Calzaferri Adv. Funct. Mater. 2001, 11, 277.b)

For the dye loading with neutral dyes, the dyes can be insertedfollowing a gas phase procedure using the double or simple ampoulemethod as described by Calzaferri et al, Chem. Eur. J. 2000, 6, 3456.This procedure also can be used for inner salt dyes like e.g. squaraines

Example 18 Protein Loading Via Adsorption and Cross-Linking

10 mg Oxazine 1 is loaded into disc-shape zeolite L (77 nm×400 nm) andis incubated with 2 mg of a polymerised digoxin antibody in a finalvolume of 1 ml MES-buffer for 15 min. For cross linking: 0.1 ml of 0.1%EDC solution is added and incubated again for 2 h. The reaction isstopped by addition of glycine following an additional incubation of 30min. After centrifugation for 30 min and resuspension a blocking stepwas performed by addition of 1 ml of 2% BSA. After several washing stepsand isolating after each washing step using centrifugation, thezeolite-antibody conjugate is stored by adding sodium azide as apreservative. Dynamic light scattering experiments shows an increase of40 nm which is in good agreement with analogue procedures done withpolystyrene particles.

Example 19 Whole Surface Modification/Functionalization of ZeoliteSurfaces

The procedure is followed as described by Huber, Calzaferri ChemPhysChem2004, 5, 239. The dye loaded and washed zeolite is dried overnight at100° C. The Oxazine-zeolite L (10 mg) is suspended in 5 ml of drytoluene containing 5 μl of aminoethylpropyl-triethoxysilane (APTES) andrefluxed for 2 h at 120° C., yielding Ox 1 zeolite L crystals havingamino groups covalently attached to the external surface. After washingthe crystals twice with toluene, they are dried again at 100° C. for 3 hand suspended in 5 ml of citrate buffer.Methyl-3-isothiocyanatopropionate is added and heated on a water bath to50-60° C. for 15 min and left at room temperature for 14 h. Aftercentrifugation and resuspension the ester function is cleaved bytreatment with a KOH solution and again centrifuged and resuspended inbuffer pH 8 for further modification.

Antibody Coupling

a) 10 mg of disc shape Ox 1 zeolite L modified with carboxylic acids arepreactivated by adding 0.1 ml of 0.1% EDC solution and treating it 20min using a roll mixer. After addition of 2 mg of a polymerised digoxinantibody in a final volume 1 ml MES-buffer the mixture is incubated for1 h.

An additional cross linking step can be performed as described inExample 18 with an additional stop reaction using glycine and blockingreaction with BSA.

b) Instead of using a two step procedure for introducing carboxylic acidgroups a one step synthesis can be done using5-(Chloro-dimethylsilyl)pentanoic acid N-hydroxysuccinimide ester(Prochimia Surfaces) and directly coupling to biomolecules without thepreactivation step as described before.

Example 20 Modification of Zeolites with Antibodies and PEG/SequentialBifunctionalization

Sequential bifunctionalization of each 10 mg dye loaded cylindrical ordisc shaped zeolite L is reached by modification of the channelentrances using FMOC-APMES synthesized according to S. Huber, G.Calzaferri, Angew. Chem. Int. Ed. 2004, 43, 6738-6742 and following theprocedure as described. The wall modification is done by using(3-Triethoxysilylpropyl)-t-butylcarbamate (ABCR Product List AB 126796)under the conditions described in example Nr. 18. The t-BOC protectinggroup can be selectively cleaved by using standard conditions (TFA,Trichloromethane). Hydrophilic PEG-NHS esters are commercially available(e.g. Sunbright®ME050HS, NOF Corporation) and are reacted with theamino-modified zeolite using a phosphate buffer pH 8.0. The FMOCprotective group is cleaved by adding the zeolite crystals to 1 ml dryDMF containing 0.2 ml piperidine. After centrifugation and washing (seeS. Huber, G. Calzaferri, Angew. Chem. Int. Ed. 2004, 43, 6738-6742) theamino groups are further modified by treating these with a high surplusof Bis-N-hydroxysuccinimido-suberate dissolved in dioxane and reactingfor 1 h. After another centrifugation and washing step thederivatizition with a biomolecule is done as described above in example19 b).

Example 21 Modification of the Whole External Surface with APTES

5 to 10 mg of zeolite L crystals were suspended in 3 mL of toluene in aPTFE tube and a big excess of approximately 1 μL of(3-aminopropyl)triethoxysilane (APTES) per mg crystals was added. Thissuspension was sonicated for 5 min and stirred for about 3 h at 110° C.After cooling down, the suspension was transferred with some tolueneinto a glass centrifugation tube and it was centrifuged. The sample waswashed afterwards with toluene, ethanol and methanol and dried in theoven at 70° C.

Example 22 Modification of the Channel Entrances with APMS

Before using the oven-dried zeolite crystals, the zeolite crystals wereleft above aqueous saturated KNO3-solution in 22% relative humidityatmosphere overnight to rehydrate. The sample was weighed to typically10-20 mg and the number of channel entrances was calculated.

To 1 mL of dry DCM in a PPCO tube were added 10 μL of(3-aminopropyl)dimethyl-methoxysilane (APMS, 0.059 mmol). To thesolution 30 mg FMOC-N-hydroxysuccinimidy-lester (FMOC-NHS, 0.089 mmol,1.5 eq.) dissolved in 1 mL of dry DCM were added dropwise and thereaction mixture was stirred at room temperature for 1.5 h to giveAPMS-FMOC. The reaction mixture was then diluted so that 10 μL of thenew solution contained the same number of the reactive APMS stopcocks asthere are channel entrances in the zeolite L sample. This amount ofdiluted solution was now added to the zeolite L suspended in 2 mL of dryn-hexane in a PTFE tube. The mixture was sonicated for 20 min to allowadsorption of the FMOC-APMS stopcock at the channel entrances andrefluxed at 70° C. for 3 h. The suspension was transferred with someadditional n-hexane into a glass centrifugation tube, it was centrifugedand the overstanding solution was pipetted off. The FMOC-APMS-zeolite Lsample was suspended in 2 mL of dry DMF containing 10% of piperidine.The deprotection was complete after stirring for 1 h at roomtemperature, giving NH2-zeolite L. The modified zeolite L sample waswashed with acetonitrile and MeOH to get rid of the remaining piperidineand dried in the oven at 70° C.

Example 23 Reaction of Zeolite L Crystals Amino Functionalised at theBases with Atto-NHS

3 to 5 mg of end modified H2N-zeolite L from Example 22 were suspendedin 1.5 mL of dry acetonitrile, one drop of triethanolamine was added andthe suspension was heated to 70° C. after sonication. An excess of Atto425-NHS ester or Atto 610-NHS ester was dissolved in 1 mL of dryacetonitrile and the solution was slowly added to the suspension and themixture was stirred for additional 1.5 h at 70° C. After cooling, thesuspension was centrifuged and the Atto-zeolite L was washed six timeswith methanol or until the supernatant showed only a weak fluorescence.

Example 24 Bifunctionalised Face Specific Crystals

The bifunctionalised crystals were synthesised from the sample that hadbeen modified at the channel entrances with Atto dyes according toExample 23. These bifunctionalised crystals were dried at mildtemperatures in the oven and were then modified on the whole externalsurface with APTES as it is described in Example 21. Afterwards, thezeolite L crystals were coloured on the entire surface by reaction ofthe all over fixed NH2-groups with Atto-NHS-ester as in Example 23(reaction in MeCN for 2 h at 70° C.).

Example 25 Bifunctionalised Mixed Crystals

3 to 5 mg of fully modified H2N-zeolite L from Example 21 were suspendedin 1.5 mL of dry acetonitrile, one drop of triethanolamine was added andthe suspension was heated to 70° C. after sonication. An excess ofequimolar amounts of Atto 425-NHS ester and Atto 610-NHS ester wasdissolved in 1 mL of dry acetonitrile and the solution was slowly addedto the suspension and the mixture was stirred for additional 1.5 h at70° C. After cooling, the suspension was centrifuged and the mixedAtto-zeolite L was washed six times with methanol or until thesupernatant showed only a weak fluorescence.

1. A conjugate comprising a zeolite labeled with a dye, a stop-cockmoiety, and covalently bound thereto an affinity binding agent.
 2. Theconjugate of claim 1, wherein the affinity binding agent is bound to astop-cock molecule.
 3. The conjugate of claim 1, wherein said affinitybinding agent is selected from the group consisting of antigen andantibody, biotin or biotin analogue and avidin or streptavidin, sugarand lectin, nucleic acid or nucleic acid analogue and complementarynucleic acid and receptor and ligand.
 4. The conjugate according toclaim 1, wherein said affinity binding agent is an antigen or anantibody.
 5. The conjugate according to any of claim 1, wherein the dyeis selected from the group consisting of a luminescent compound; afluorescent compound; a light absorbing compound; a radioactive compoundthat can be visualized by photographic plates, a metal compound that canbe visualized using x-rays; a compound that can be visualized usingmagnetic resonance imaging (MRI); an isotope that can be imaged by x-raycomputed tomography (CT), ultrasound or photon emission computedtomography (SPECT).
 6. The conjugate of claim 5, wherein said dye isselected from the group consisting of a luminescent compound; afluorescent compound; and a radioactive compound.
 7. The conjugate ofclaim 5, wherein said dye is a dye is visualizable by magnetic resonanceimaging (MRI), x-ray computer tomography (CT) or ultrasound or photonemission computed tomography (SPECT).
 8. A method for producing aconjugate comprising: a) labeling a zeolite having channels with a dyeto form a dye-labeled zeolite b) closing the channels by a stop-cockmolecule c) adding an affinity binding agent and d) using an appropriatecoupling chemistry to form a covalent linkage between the zeolite andthe affinity binding agent.
 9. A method for measuring an analyte by anin vitro method, the method comprising the steps of a) providing asample suspected or known to comprise the analyte b) contacting saidsample with a conjugate of a dye-labeled zeolite and an affinity bindingagent under conditions appropriate for formation of an analyte-conjugatecomplex, c) measuring the complex formed in step (b) and therebyobtaining a measure of the analyte.
 10. A labeled zeolite loaded with animageable dye, wherein the imageable dye is selected from the groupconsisting of a metal compound that can be visualized using x-rays; acompound that can be visualized using magnetic resonance imaging (MRI);an isotope that can be imaged by x-ray computed tomography (CT),ultrasound or photon emission computed tomography (SPECT).
 11. Thelabeled zeolite according to claim 10, wherein said imageable dye is adye that can be visualized by MRI, CT or SPECT.
 12. The labeled zeoliteaccording to claim 10, wherein the labeled zeolite is zeolite L crystal.13. A carboxyester functionalized zeolite crystal.
 14. The carboxyesterfunctionalized zeolite crystal according to claim 13, whereas saidzeolite crystal is labeled with a dye.
 15. The carboxyesterfunctionalized zeolite crystal according to claim 14, whereas the dye isselected from the group consisting of a fluorescent compound; aluminescent compound; a radioactive compound that can be visualized byphotographic plates, a metal compound that can be visualized usingx-rays; a compound that can be visualized using magnetic resonanceimaging (MRI); an isotope that can be imaged by x-ray computedtomography (CT), ultrasound or photon emission computed tomography(SPECT).